Modified cytotoxic tall cell line and compositions and methods for manufacture and use thereof as therapeutic reagents for cancer

ABSTRACT

The invention provides a modified human cytotoxic T cell line TALL-104, which is characterized by dual activity in vitro and in vivo against tumor cells and virus-infected cells. Also provided are effective and safe methods for use of the modified TALL-104 cells in adoptive therapy of cancer and untreatable viral diseases in MHC-mismatched recipients, and in marrow purging to achieve complete eradication of residual tumor cells from marrows of patients with leukemia and other types of cancer. Also provided are effective and safe methods for use of the modified TALL-104 cells in the manufacture of a veterinary composition for adoptive therapy of canine lymphoma and feline leukemias.

This invention has been made with the financial assistance of GrantsCA-47589, CA-10815, and CA-42232 from the National Institutes of Health.The United States government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of International Patent Application No.PCT/US94/05374, filed May 12, 1994, which is a continuation-in-part ofU.S. patent application Ser. No. 08/063,188, filed May 14, 1993, nowabandoned which was a continuation-in-part of U.S. patent applicationSer. No. 07/859,927, filed Mar. 30, 1992, now U.S. Pat. No. 5,272,082.These parent applications are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates generally to the modified cytotoxic Tlymphoblastic leukemia (T-ALL) cell line and its therapeutic uses.

BACKGROUND OF THE INVENTION

Cytolytic T lymphocytes (CTL) and natural killer (NK) cells havecomplementary roles in providing defense against tumor cells,virus-infected cells and other invading agents see, e.g., Oycius andYoung, Cancer Cells 2:138 (1990)!. CTL are CD3⁺ /T-cell receptor(TCR)αβ⁺, CD8⁺, CD16⁻ cells which recognize specific peptides inassociation with class I molecules of the major histocompatibilitycomplex (MHC). NK cells are CD3⁻ /TCR⁻, CD16⁺, CD56⁺ cells which areconstitutively able to lyse virus-infected cells and some tumor celllines (but not fresh tumor cells) without prior sensitization andwithout restriction by MHC antigens. The molecules specifically involvedin target cell recognition by NK cells (the putative NK receptor) are asyet unknown.

Over the past decade, lymphokine-activated killer (LAK) activity hasbeen defined as an additional type of MHC non-restricted killing. LAKcells are conventionally generated upon short-term culture of humanperipheral blood cells in interleukin-2 (IL-2) and possess phenotypiccharacteristics of either NK (CD3⁻ CD16⁺) or T (CD3⁺ CD16⁻) cells, thusconstituting an heterogeneous (mixed) cell population. LAK cells areable to kill NK-resistant tumor cell lines, autologous and allogeneicfresh tumor targets, and virus-infected cells while remainingsubstantially non-toxic to normal cells E. Grimm et al., J. Exp. Med.,144:1823 (1982); S. Rosenberg, J. Natl. Can. Inst., 75:595 (1985)!.

A. Adoptive Immunotherapy of Cancer Using LAK Cells and CTL

The use of LAK cells as anti-cancer effectors in combination withrecombinant human (rh) IL-2 in adoptive immunotherapy of cancer hasresulted in the achievement of temporary remission in a variety of humancancers, especially renal carcinoma and melanoma S. A. Rosenberg et al.,New Engl. J. Med., 313:1485 (1985); S. A. Rosenberg et al., New Engl. J.Med., 316:889 (1987); W. H. West et al., New Engl. J. Med., 316:898(1987)!. However, LAK cell therapy has had little success againstestablished metastatic disease Rosenberg et al, (1987), cited above!.Two major problems affect the efficiency of adoptively transferred LAKcells: one is the inadequate tumoricidal activity of these MHCnon-restricted polyclonal killer cells; the second is the unavailabilityof sufficient numbers of activated effectors that retain both cytotoxicand tumor targeting capabilities. The high doses of rh IL-2 administeredto the patients to circumvent these limitations have been associatedwith significant toxicity and adverse side effects Rosenberg et al.,(1985) and Rosenberg et al., (1987), both cited above!.

Several alternate approaches have been proposed to improve cancertherapy, including the use of antigen-specific tumor infiltratingcytotoxic lymphocytes (TIL) Itoh et al, Cancer Res., 46:3011 (1986) andMuul et al, J. Immunol., 138:989 (1987)!, the use of LAK cells linkedwith antibodies to tumor cell surface antigens Takahashi et al, Science,259:1460 (1993) and Nistico' et al, J. Clin. Invest., 90:1093 (1992)!,and genetic engineering of tumor cells with cytokine genes (such asIL-2, TNF, IFN-γ, IL-4, IL-6, and IL-7), to promote enhanced anti-tumorimmunity Gansbacher et al, J. Exp. Med., 172:1217 (1990); Gansbacher etal, Cancer Res., 50:7820 (1990); Hock et al, J. Exp. Med., 174:1291(1991); Golumbeck et al, Science, 254:713 (1991); and Asher et al, J.Immunol., 146:3227 (1991)!. In particular, TIL expanded from tumorinfiltrates in the presence of high doses of rh IL-2 (6000 U/ml) growrapidly and become potent cytolytic anti-tumor effectors Itoh, et al.,Cancer Res. 46:3011 (1986); Rosenberg, S. A., Ann. Surg. 208:121 (1988)!which can be transferred back into the tumor-bearing individuals(together with high doses of IL-2), where they migrate into tumorlesions and promote tumor regression. However, IL-2 therapy incombination with these specific CTL (TIL/IL-2 therapy) has yieldedobjective tumor regression in only 20-30% of patients with melanoma andcarcinoma of the kidney Rosenberg, et at, Ann Surg. 210:474 (1989)!. Inaddition, high-dose cytokine-related toxicities (including the capillaryleak syndrome and renal dysfunction) have precluded extended therapiesin many patients Rosenberg et al, (1987), cited above; Rosenberg, et al,Ann. Surg. 210:474 (1989); Lee, et al, J. Clin. Oncol. 7:7 (1989); andLotze, et al. Hum. Immunol. 28:198 (1990)!.

Bone Marrow Purging and Consolidative Immunotherapy (LAK/IL-2 Therapy)in Patient's with Leukemia

Autologous bone marrow transplantation (ABMT) has shown preliminarypromising results both in children with acute myeloblastic leukemia(AML) and acute lymphoblastic leukemia (ALL) in second remission, and inadults with AML in first remission or ALL in second remission Govin etal, Bone Marrow Transpl., 4:1206 (1989)!. Ex vivo treatment of marrowswith pharmacological agents (e.g., 4-hydroperoxycyclophosphamide or4-HC), or appropriate monoclonal antibodies to remove residual leukemiccells, seems to contribute to the efficacy of these autografts, andmarrow purging has been incorporated into several of these studiesKaizer et al, Blood, 77:1534 (1985)!. However, despite the use of thesepurging procedures, the relapse rate in leukemic patients after ABMT isstill almost 50% Govin et al, (1989), cited above!. Improved marrowpurging protocols are, therefore, needed to achieve total eradication ofnon-detectable malignant blasts in order to prevent disease recurrence.

Within hematologic malignancies, therapy with LAK cells and IL-2 hasinduced some clinical responses in patients with advanced malignantlymphoma or AML. It is postulated that administration of LAK cells withIL-2 might prevent or delay relapses if used as consolidativeimmunotherapy against the minimal residual disease (MRD) which existsafter ABMT. Although IL-2-responsive LAK precursor cells are in thecirculation as early as 3 weeks after ABMT, full LAK activity isrecovered only 70-80 days after the transplant. The feasibility ofgenerating and administering autologous LAK cells and IL-2 after ABMT inpatients with high risk of relapse is now under investigation. However,this therapeutic modality might have practical limitations in manypatients with AML or ALL due to the scarcity of cytotoxic cells in theirblood and to the often rapid disease progression.

C. Treatment of Viral Infections with CTL

Dr. P. Greenberg's group (Fred Hutchinson Cancer Research Center,Seattle, Wash.), have been adoptively transferring CTL (CD3⁺ CD8⁺)specific for cytomegalovirus (CMV) to reconstitute CMV-specific immunityin immunosuppressed patients S. A. Riddell et al, In Vitro Devel. Biol.,28:76A (1992)!. Their protocol includes obtaining T cells from theperipheral blood of the patients, expanding them to large quantities invitro in the presence of virus-infected cells and IL-2, and geneticallyengineering the specific CTL so generated with a "suicide" gene (herpesvirus thymidine kinase). Two problems are inherent to the adoptiveimmunotherapy protocol with virus-specific CTL clones. First, thecytotoxic cells must be derived from the same patient in which they willbe transferred back for treatment. Second, a retroviral mediated genetransfer must be used to introduce in the CTL clones a selectable markergene and a "suicide" gene to increase the safety of therapy in thissetting and eliminate the virus-specific CTL, in case the clonesstimulate a life-threatening inflammatory immune response in thepatient.

Thus, in the application of adoptive cellular immunotherapy againsttumors or against vital-diseases, the therapy by necessity requiresobtaining peripheral blood lymphocytes from individual patients, growingthem in vitro for an adequate length of time in order to expand apopulation of IL-2-activated killer cells (LAK or CTL), and reinjectingsuch cells into the same patient.

There remains a need in the art for convenient, safe, and more effectivemethods and compositions for the adoptive immunotherapy of cancer anduntreatable viral infections, and for purging residual malignant cellsremaining after cytoreductive treatments in the bone marrow of patientswith leukemia.

SUMMARY OF THE INVENTION

In response to the above-stated need in the art, the present inventionprovides a clonal, MHC non-restricted and immortal effector cellpopulation able to recognize and attack selectively tumor andvirus-infected cells; this effector cell is unique as it represents auniversal and powerful reagent for cancer therapy and for treatment ofviral diseases.

In one aspect of this invention, a method for modifying an established(immortal) cell line, designated TALL-104, which displays potent MHCnon-restricted cytotoxicity against a broad spectrum of tumor cells andvirus-infected cells, without damaging or killing cells from normaltissues, is disclosed. This method modifies the TALL-104 cells to conferone or more desired characteristics that will make the cells suitablefor use in marrow purging and in adoptive transfer immunotherapy inallogeneic (HLA-mismatched) recipients. This method includes the stepsof maximizing the cytotoxicity of the TALL-104 cell line by a shorttreatment (18 hours) in vitro with a selected cytokine, e.g., IL-2 aloneor combined with IL-12, followed by γ-irradiating the activated cells toarrest irreversibly their proliferation.

As another aspect, the invention provides the modified TALL-104 cellline prepared by the above-described method.

As a further aspect, the invention provides a method for killing tumorcells (or virus-infected cells) in vivo. The method involvesadministering to a patient, e.g., a immunosuppressed recipient, themodified cytotoxic TALL-104 cell line described above. The modified cellline is preferably administered by i.v. injection in a suitable carrier,such as saline.

As still a further aspect, the invention provides a method for killinglymphomas and leukemias in animals, particularly domestic animals suchas dogs and cats, respectively. The method involves administering to animmunosuppressed animal the modified cytotoxic TALL-104 cell linedescribed above, preferably by i.v. injection in a suitable carrier,such as saline.

In yet another aspect, the invention provides a method for eliminatingresidual tumor cells from patients' bone marrow (marrow purging) by exvivo treatment of such marrow with the modified cytotoxic TALL-104 cellline followed by autologous bone marrow transplant (ABMT) and byconsolidative immunotherapy with the modified TALL-104 cells.

Other aspects and advantages of the present invention are discussedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a bar graph demonstrating the cytotoxic activity (expressedas 40% lytic units (LU)) of IL-2 activated TALL-104 cells against HIVchronically infected (U-1) and uninfected (U937) myelomonocyticleukemia. Phorbol myristate acetate (PMA) was given to the targets 24and 48 hours before the cytotoxic assay to induce virus production.

FIG. 1B is a bar graph demonstrating the cytotoxic activity of IL-2activated TALL-104 cells against HIV chronically infected (AcH₂) anduninfected (CEM) T lymphocytic leukemia. Treatment of targets with PMAfor 24 and 48 hours increased viral production and sensitivity to lysisby TALL-104 cells.

FIG. 2A is a graph illustrating the dose-dependent effects ofcyclosporin A (CsA) on the cytotoxicity (⁵¹ Cr release) of IL-2activated TALL-104 cells.

FIG. 2B is a graph illustrating the dose- dependent effects ofcyclosporin A (CsA) on the cytotoxicity (⁵¹ Cr release) of IL-2activated LAK cells.

FIG. 2C is a bar graph illustrating the dose-dependent effects of CsA onthe proliferation (³ H-TdR uptake) of IL-2 activated TALL-104 cells andLAK cells. The effectors were treated for 18 hours with CsA before beingtested for ³ H!TdR uptake and cytotoxic activity against Raji cells (anNK-resistant target). Results of one representative experiment areshown; similar data were obtained with LAK cells from three donors andwith TALL-104 cells in three more experiments.

FIG. 3 illustrates the comparative biodistribution of irradiated (i.e.,modified) and non-irradiated TALL-104 cells in SCID mouse tissues.Detection of the intraperitoneally (i.p.) implanted TALL-104 cells wasdone by PCR using human ALU-DNA sequences.

FIG. 4A is a bar graph illustrating the long-term effects ofγ-irradiation (4000 rads) on the cytotoxic efficiency of TALL-104 cells.The effectors were tested for ability to lyse ⁵¹ Cr-labeled Raji cellson days 1, 4, and 7 after γ-irradiation; IL-2 (100 U/ml) was added tothe effectors the day before each cytotoxic assay in order to boostertheir lytic activity. Results are expressed as 40% LU. The same cellswere tested for ³ H!TdR uptake. Results are representative of oneexperiment out of three performed with different LAK donors with similarresults.

FIG. 4B is a bar graph illustrating the long- term effects ofγ-irradiation (4000 rads) on the cytotoxic efficiency of LAK cells. Theeffectors were tested and results obtained as described for FIG. 4A.

FIG. 4C is a bar graph illustrating the long-term effects ofγ-irradiation (4000 rads) on the proliferation of TALL-104 cells. Theeffectors were tested and results obtained as described for FIG. 4A.

FIG. 4D is a bar graph illustrating the long-term effects ofγ-irradiation (4000 rads) on the proliferation of LAK cells. Theeffectors were tested and results obtained as described for FIG. 4A.

FIG. 5A is a graph illustrating the short-term effects of γ-irradiation(4000 rads) on the tumoricidal activity of TALL-104 cells. The effectorswere tested immediately after irradiation against the U937myelomonocytic leukemia tumor cell line in a 4 hour ⁵¹ Cr-release assay,as compared to non-irradiated effectors. Mean±SD values for LAK cellswere calculated from three experiments in which three different LAKdonors were used. No SD were observed in the case of modified orunmodified TALL-104 cells in several cytotoxic assays.

FIG. 5B is a graph illustrating the short-term effects of γ-irradiation(4000 rads) on the tumoricidal activity of LAK cells. The effectors weretested immediately after irradiation against the U937 myelomonocyticleukemia tumor cell line in a 4 hour ⁵¹ Cr-release assay, as compared tonon-irradiated effectors. Mean±SD values were calculated as described inFIG. 5A.

FIG. 5C is a graph illustrating the short-term effects of γ-irradiation(4000 rads) on the tumoricidal activity of TALL-104 cells. The effectorswere tested immediately after irradiation against the U-87MGglioblastoma tumor cell line in a 4 hour ⁵¹ Cr-release assay, ascompared to non-irradiated effectors. Mean±SD values were calculated asdescribed in FIG. 5A.

FIG. 5D is a graph illustrating the short-term effects of γ-irradiation(4000 rads) on the tumoricidal activity of LAK cells. The effectors weretested immediately after irradiation against the U-87MG glioblastomatumor cell line in a 4 hour ⁵¹ Cr-release assay, as compared tonon-irradiated effectors. Mean±SD values were calculated as described inFIG. 5A.

FIG. 5E is a graph illustrating the short-term effects of γ-irradiation(4000 rads) on the tumoricidal activity of TALL-104 cells. The effectorswere tested immediately after irradiation against the 2774 ovariancarcinoma tumor cell line in a 4 hour ⁵¹ Cr-release assay, as comparedto non-irradiated effectors. Mean±SD values were calculated as describedin FIG. 5A.

FIG. 5F is a graph illustrating the short-term effects of γ-irradiation(4000 rads) on the tumoricidal activity of LAK cells. The effectors weretested immediately after irradiation against the 2774 ovarian carcinomatumor cell line in a 4 hour ⁵¹ Cr-release assay, as compared tonon-irradiated effectors. Mean±SD values were calculated as described inFIG. 5A.

FIG. 6 is a bar graph illustrating the ability of γ-irradiated(modified) and unmodified TALL-104 cells to selectively kill tumortargets and not normal cells. ⁵¹ Cr-labeled tumor targets (see Table IIIand IV), normal bone marrow (BM) cells (BM1-BM3) and normal melanocytes,all listed in the ordinate were tested (10⁴ /well) for susceptibility tolysis by modified TALL-104 cells at four different concentrations. Theresults of the 4 hour cytotoxic assay were expressed as 30% LU/10⁸effectors.

FIG. 7A is a bar graph illustrating the lack of toxicity of γ-irradiated(modified) TALL-104 cells against bone marrow precursors, as determinedby their capacity to form colonies in methylcellulose. Modified TALL-104cells were mixed with BM cells from Donor A for 4 hours at the indicatedratios. The cell mixtures were subjected to a clonogenic assay and thecolonies (CFU or BFU) counted after 14 days.

FIG. 7B is a bar graph illustrating the results of the process describedfor FIG. 7A, but using BM cells from a second Donor B.

FIG. 7C is a bar graph illustrating the results of the process describedfor FIG. 7A, except that the BM and TALL-104 cells were mixed for 18hours.

FIG. 7D is a bar graph illustrating the results of the process describedfor FIG. 7C, but using BM cells from a second Donor B.

FIG. 8 is a gel illustrating the efficiency of modified TALL-104 cellsin purging a bone marrow of leukemic cells (in this case the U937 cellline tagged with the Neomycin-resistance gene (Neo⁺ U937 cells). BMcells from a normal donor were mixed with different number of Neo⁺ U937cells as indicated. IL-2/IL-12 activated, γirradiated (modified)TALL-104 cells were added to the mixture for 18 hours in the presence of10 U/ml of DNAse I. Cellular DNA was extracted and subjected to PCRamplification using neomycin-resistant gene specific primers. Anoligonucleotide probe specific for the amplified sequences was used todemonstrate the specificity of the PCR products.

FIG. 9 is a graph illustrating the survival of U937-engrafted SCID miceafter treatment with IL-2 activated, non-irradiated TALL-104 cells. Micewere injected i.p. with 10⁷ U937 cells. TALL-104 cells, activated exvivo with 100 U/ml rh IL-2, were injected i.p. (2×10⁷ cells) at variousintervals, as indicated. rh IL-12 was given to one group of mice i.p.for 7 days at the dose of 1 μg/day.

FIG. 10A is a graph illustrating the survival of SCID mice engraftedi.p. with neomycin-tagged U937 cells and treated with modified(γ-irradiated) TALL-104 cells. Mice received 10⁷ U937 cells on day 0,and modified TALL-104 cells (2×10⁷) three times on alternate days,starting from the day of tumor challenge. Both control (U937 injected)and experimental (U937/TALL-104 injected) mice received IL-2 or IL-12daily for 1 week.

FIG. 10B is a gel illustrating the PCR detection (as obtained on gels)of residual AML in SCID mice upon adoptive transfer of modified(γ-irradiated) TALL-104 cells. Mice received U937 and modified TALL-104cells and cytokines, as outlined above in FIG. 10A. Cellular DNA wasextracted on day 55 from the BM of asymptomatic animals and subjected toPCR amplification using primers specific for both human ALU sequencesand the neomycin resistance gene. DNAs were visualized with ethidiumbromide after electrophoresis in 2% agarose gels. Southern blots werehybridized with the indicated oligo probes. Lanes 1 and 5 represent thenegative (water) and positive (neomycin⁺ U937 cells) controls,respectively. Lanes 2-4 represent the experimental mice, which receivedboth U937 and γ-irradiated TALL-104 cells, in conjunction or not withcytokines.

FIG. 11A is a graph showing the ability of modified (IL-2 activated)TALL-104 cells to lyse cat leukemia (Cat 3281 and Cat 3201B) cell linesand dog lymphoma (Pilgrim and Tertiat's) cell lines in a 4 hour ⁵¹ Crrelease assay using various E/T ratios.

FIG. 11B is a graph showing the ability of modified (IL-2 activated)TALL-104 cells to lyse cat leukemia (Cat 3281 and Cat 3201B) cell linesand dog lymphoma (Pilgrim and Tertiat's) cell lines in a 18 hour ⁵¹ Crrelease assay using various E/T ratios.

FIG. 12A is a graph demonstrating the ability of modified (IL-2activated, lethally irradiated) (▪) TALL-104 cells to lyse a doglymphoma (Pilgrim) cell line with similar efficiencies as unirradiated(∘) TALL-104 cells. The ⁵¹ Cr release assays were done for 4 hours atvarious E/T ratios.

FIG. 12B is a graph demonstrating the ability of modified (IL-2activated, lethally irradiated) (▪) TALL-104 cells to lyse a doglymphoma (Pilgrim) cell line with similar efficiencies as unirradiated(∘) TALL-104 cells. The ⁵¹ Cr release assays were done for 18 hours atvarious E/T ratios.

DETAILED DESCRIPTION OF THE INVENTION

The present invention meets the above-mentioned need in the art byproviding a method for modifying a highly cytotoxic T cell line(TALL-104) established from a child with T lineage acute lymphoblasticleukemia, the modified TALL-104 cell line as therapeutic reagents. TheTALL-104 cell line is immortal (i.e., can be maintained indefinitely invitro). The unmodified cell line is believed to be the first immortalhuman cell line having such a broad and extremely high antiviral andantitumor cytotoxic activity in vitro and in vivo, as described below.The growth of these modified cells can be arrested when used in vivo foradoptive transfer therapy, and the modified cells would not be rejectedby immunosuppressed HLA-mismatched recipients. In addition, thesenon-proliferating cells maintain a high migratory ability in the tissuesof the immunosuppressed recipient, thus being able to interact with andkill tumor and virus-infected targets.

A. The Unmodified TALL-104 Cell Line

The isolation, characterization, and maintenance of unmodified TALL-104cells are described in detail in Examples 1 and 2 see, also, O'Connor etal., Blood, 77:1534-1545 (1991), which is incorporated by referenceherein!. See, also, U.S. Pat. No. 5,272,082, issued Dec. 21, 1993.

The unmodified TALL-104 cell line is permanently established in tissueculture and has been continuously maintained in vitro since 1990. Thecell line is kept within the laboratory of Dr. Daniela Santoli at TheWistar Institute of Anatomy and Biology, Philadelphia, Pa., and has beendeposited with American Type Culture Collection (ATCC) as ATCC AccessionNo. CRL 11386.

The unmodified TALL-104 T cell line of this invention, designatedTALL-104, has a CD3⁺ /TCRαβ⁺, CD4⁻, CD8⁺, CD56⁺, CD16⁻ phenotype, asdefined by immunofluorescence analysis with a panel of monoclonalantibodies and compared to the phenotype of purified Natural Killer (NK)cells and lymphokine-activated killer (LAK) cells (see Table I below).

                  TABLE I                                                         ______________________________________                                        Surface markers                                                                           TALL-104     LAK     NK                                           ______________________________________                                        CD2         39-45.sup.a  79-94   80-90                                        CD3          98-100      78-95   <1                                           TCR-αβ                                                                         98           45-53   <1                                           CD4         <1           44-62   <1                                           CD8         91-96        37-55   60-80                                        CD16        <1           10-20    99                                          CD56         94           9-17    99                                          ______________________________________                                         .sup.a Percent positive cells, as determined by immunofluoresence             analysis.                                                                

These surface markers indicate that TALL-104 cells have a phenotypecompatible with a clonal cytotoxic T cell subset. The lack of surfaceexpression of CD16, a classical surface marker of NK cells, and thepresence of a T cell receptor (TCR) with the α/β heterodimer, bothindicate that these cells belong to the T cell lineage and not to the NKcell lineage. By contrast, the antigenic profile of IL-2-expanded LAKcells, as shown above, indicates the heterogeneity of the LAK cellpopulation which includes activated NK and T cells.

This TALL cell line recognizes the same tumor targets as conventionalLAK cells but, being clonal, it is much more potent. In fact, aftermodification as described herein in part B below, (e.g., after lethalirradiation (4000 rads) using a Cesium source) the TALL-104 cells, butnot LAK cells, remain highly cytotoxic. This phenomenon is the oppositeof the one that has been reported in the literature with primarycytotoxic leukemic T cell cultures, where irradiation abrogatedcompletely their cytotoxic activity in response to IL-2 Kaufmann et al.J. Immunol. 139:977-982, (1987) !.

The TALL-104 cell line has a characteristic chromosomal translocationinvolving chromosomes 11 and 14, i.e., at t(11;14) (p13:q11). TALL-104cell line is further characterized by TCR rearrangements alpha, beta,gamma and delta, and the ability to produce lymphokines such as GM-CSF,IFN-gamma, TNF-α, and TGF-β1.

The TALL-104 cell line is extremely cytotoxic against a broad spectrumof tumor cells and of virus-infected cells. Specifically, the cell linedisplays potent MHC non-restricted cytotoxicity against a broad varietyof tumor cells (including human leukemias and solid tumors) (Tables IIand III) and virus-infected cells (Table IV and FIG. 1A and B), withoutdamaging or killing cells from normal tissues. Tables II and III alsoshow that conventional NK and LAK cells display a lower cytotoxicefficiency and a more restricted spectrum of activity against tumor andvirus-infected targets, as compared to the TALL-104 cell line.

Importantly, as shown in Table II below, the TALL-104 cell line iscompletely resistant to lysis by allogeneic NK and LAK cells and CTL,and thus would not be destroyed by the immune system of HLA-mismatchedrecipients. Cytotoxic activity was measured as described in Cesano andSantoli, In Vitro Cell Dev. Biol., 28A:648-656 (1992).

                  TABLE II                                                        ______________________________________                                        Cytotoxic efficiency of TALL-104 cells as compared to                         normal NK and LAK cells against leukemic targets                                         Effectors                                                          Leukemic target                                                                            NK.sup.a   LAB.sup.b  TALL-104.sup.c                             ______________________________________                                        Cell Lines                                                                    K562 (CML)   .sup. 2125 ± 1125.sup.d                                                               7125 ± 871                                                                            7831 ± 112                              U937         2373 ± 915                                                                             6908 ± 1002                                                                          7183 ± 82                               (myelomonocytic)                                                              THP-1 (monocytic)                                                                          <1         2113 ± 307                                                                            2983 ± 112                              HL60         308 ± 115                                                                             4168 ± 605                                                                            5999 ± 99                               (promyelocytic)                                                               Raji (B-lymphoma)                                                                          <1         1160 ± 302                                                                            3890 ± 201                              Daudi (B-lymphoma)                                                                         102 ± 13                                                                              3987 ± 731                                                                            7999 ± 81                               ALL-1 (Pre-B ALL)                                                                          <1         113 ± 32                                                                              1983                                       ALL-2 (Pre-B ALL)                                                                          <1          83 ± 31                                                                              2001                                       ALL-3 (Pre-B ALL)                                                                          <1         201 ± 83                                                                              1876                                       TALL-101 (T-ALL)                                                                           <1         1152 ± 102                                                                            2931                                       TALL-103/2 (T-ALL)                                                                         <1         820 ± 98                                                                              3783                                       TALL-103/3 (T-ALL)                                                                         280 ± 80                                                                              1476 ± 123                                                                            5676                                       TALL-104 (T-ALL)                                                                           <1         <1         <1                                         TALL-105 (T-ALL)                                                                           <1         360 ± 56                                                                              1176                                       TALL-106 (T-ALL)                                                                           <1         1120 ± 132                                                                            4987                                       Primary                                                                       leukemic samples                                                              AML (FAB-M5) <1         <1         3183                                       T-ALL        <1         <1         1183                                       Normal bone marrows                                                           Donor A      <1         <1         <1                                         Donor B      <1         <1         <1                                         Donor C      <1         <1         <1                                         ______________________________________                                         .sup.a Freshly isolated PBL from healthy donors.                              .sup.b PBL from healthy donors incubated in rh IL2 (100 U/ml) for 3 days.     .sup.c TALL104 cells maintained in rh IL2 (100 U/ml).                         .sup.d 40% LU/10.sup.8 cells.                                                 .sup.e Not determined.                                                   

                  TABLE III                                                       ______________________________________                                        Cytotoxic efficiency of TALL-104 cells as compared to                         normal NK and LAK cells against cell lines derived from solid tumors                    Effectors                                                           Solid tumor target                                                                        NK.sup.a  LAK.sup.b   TALL-104.sup.c                              ______________________________________                                        Glioblastoma                                                                  U373-MG     .sup. <1.sup.d                                                                           2777 ± 1326                                                                           4186                                        U87-MG      <1        4183 ± 493                                                                             8706 ± 210                               WA2323-2    <1        1008 ± 375                                                                             4121                                        Neuroblastoma                                                                 CHP-100     .sup. n.d..sup.e                                                                        860         3383                                        CHP-134     n.d.      998         1943                                        Melanoma                                                                      FO-1        429 ± 171                                                                            2073 ± 27                                                                              7108                                        FO-1 (HLA.sup.-  variant)                                                                 775 ± 550                                                                            2680 ± 385                                                                             7992                                        SK-MEL-33   388 ± 15                                                                             1700 ± 101                                                                             6899                                        SK-MEL-33   667 ± 480                                                                            1916 ± 290                                                                             8193                                        (HLA.sup.-  variant)                                                          WM-451      <1        2633 ± 789                                                                             3158                                        WM-1985     <1        2083 ± 535                                                                             4165                                        WM-793      <1        1924 ± 549                                                                             8291                                        WM-852      <1        2151 ± 347                                                                             8970                                        Bladder carcinoma                                                             5637        <1        5086 ± 240                                                                             6177                                        Ovarian carcinoma                                                             OVCA3       <1        n.d.        4848                                        2774        <1        n.d.        2177 ± 83                                NIH-OVCA3                         2369                                        Breast carcinoma                                                              H.sub.s 568-T                                                                             n.d.      n.d.        6522 ± 121                               MDA-MD468   n.d.      n.d.        7198                                        Prostate carcinoma                                                            PC-3        n.d.      n.d.        5382                                        DU-145      n.d.      n.d.        8893                                        Ln CHP      n.d.      n.d.        2144                                        Lung carcinoma                                                                A459        <1         212 ± 143                                                                             1598 ± 113                               CaLu-1      <1         316 ± 215                                                                             2264                                        CaLu-6      <1        161 ± 23 1239                                        ______________________________________                                         .sup.a Freshly isolated PBL from healthy donors.                              .sup.b PBL from healthy donors incubated in rh IL2 (100 U/ml) for 3 days.     .sup.c TALL104 cells maintained in rh IL2 (100 U/ml).                         .sup.d 40% LU/10.sup.8 cells.                                                 .sup.e Not determined.                                                   

Further, as illustrated in Table IV below, the cytotoxic activity of theTALL-104 cell line against virus-infected cells is also higher than thatof NK and LAK cells from healthy donors measured as described in Santoliet al, J, Immunol., 121:526-531 (1978) and J. Immunol., 121:532-538(1978), incorporated by reference herein. The results are expressed in40% lytic units (LU)/10⁸ cells. Interestingly, at the effector-target(E/T) ratio of 50:1, HIV-infected CEM and U937 cells were killed asefficiently as the uninfected counterpart by purified NK cells (TableIV). In contrast, at the E/T ratio of 10:1, TALL-104 cells killed moreefficiently the HIV infected targets than the uninfected ones. These invitro results indicate the ability of the TALL effectors to lyseefficiently virus-infected targets and the utility of the TALL cells inthe treatment of lethal vital diseases in vivo.

It is also anticipated that when modified as described in part B (i.e.,lethally irradiated and cytokine activated) TALL-104 cells can beemployed to treat non-human primates (cynomologous monkeys) infectedwith simian immunodeficiency virus (SIV), which is the equivalent ofHIV. This is done by administering the modified TALL-104 cells intoimmunosuppressed monkeys at various stages of SIV infection. Suchstudies permit evaluation of the possible toxicity of TALL-104 cells inprimates. If toxicity develops due to release by TALL-104 cells of highlevels of toxic mediators, such as TNF-α, TNF receptors may beeliminated from the TALL-104 cells by conventional methods, such as theconstruction of deletion mutants (see U.S. Pat. No. 5,272,082, issuedDec. 21, 1993). Such evaluation permits the development of safe,non-toxic protocols of adoptive transfer therapy in humans and animals.

                  TABLE IV                                                        ______________________________________                                        Cytotoxic efficiency of γ-irradiated TALL-104 cells                     against virus-infected cells                                                  Target        Virus  NK       LAK  TALL-104                                   ______________________________________                                        FS-5 (fibroblasts)                                                                          none   <1       110  299                                                      CMV    810      2683 10,183                                     CHP-134       none   ND       ND   1043                                       (neuroblastoma)                                                                             PR8    ND       ND   7407                                       CEM (leukemic none   111      1483 1692                                       T cell line)  HIV    155      1603 6327                                       U937 (leukemic                                                                              none   746      1382 4462                                       myeloid cell line)                                                                          HIV    697      2129 8737                                       ______________________________________                                         CMV = cytomegalovirus                                                         PR8 = influenza virus                                                         HIV = human immunodeficiency virus                                            ND = not determined                                                      

The cytotoxic properties of the TALL-104 cell line are described in moredetail in Cesano and Santoli, In Vitro Cell Dev. Bio. 28A:648 (1992);and Cesano, et al, J. Immunol., 151:2943 (1993). The above referencesare incorporated by reference herein for this non-essential disclosure.

The cell line of this invention is further characterized by being freeof any contaminating virus, including Epstein-Bart virus (EBV) andretroviruses such as HTLV-1, HTLV-II, and HIV, as tested byelectronmicroscopy, reverse transcriptase assays and Southern blotanalysis using specific viral DNA probes.

The TALL-104 cell line offers the advantage over conventional LAK and NKcells from healthy donors and primary leukemic T cell cultures of beingimmortal, i.e., permanently and rapidly growing in vitro in the presenceof rhIL-2, and thus providing unlimited material for in vitro and invivo studies. Another advantage of these cells is that they are clonalpopulations, thus phenotypically and functionally stable therebyeliminating the intervariability and reproducibility problemsencountered with LAK cells from different donors which representheterogenous cell populations.

A further advantage of the TALL-104 cell line is that it retainscytotoxic activity even in the presence of immunosuppressive drugs,e.g., the drugs used in organ transplantation like Cyclosporin A (CsA)that block the immune response of the host. As shown in FIG. 2A-C, evenwhen used at doses 3 logs higher than the therapeutic levels reached inthe patients blood (e.g. 100 μg/ml), CsA does not impair the cytotoxicactivity of the TALL-104 cells. In contrast, CsA impairs thecytotoxicity of LAK cells (FIG. 2A-C).

Based on the unique behavior of TALL-104 cells in the presence of asuitable immunosuppressive drug, such as CsA, these cells can beinjected in MHC mismatched immunosuppressed recipients, and still exerttheir cytotoxic activity without being rejected. It is anticipated thatthese cells will display the same activity when administered with, orexposed to, other immunosuppressive drugs routinely used in clinicalpractice (e.g., glucocorticoids).

Unmodified TALL-104 cells can grow in vivo in severe combinedimmunodeficient (SCID) mice infiltrating their tissues Cesano et al.Blood, 77:2463-2474 (1991)!. These cells do not compartmentalize inspecific tissues but rather can reach every tissue in the organismdepending on the route of administration. Because the cell line might beleukemogenic if injected into an immunosuppressed host, it must bemodified to control or arrest cell growth without affecting its killerfunction.

B. Modified TALL-104 Cell Line

The modification of TALL-104 cells according to the invention and asdescribed in detail below involves both cytokine potentiation of thelytic function and growth arrest by lethal irradiation. Suchmodification makes the cells suitable (effective and safe) for use inmarrow purging to achieve complete eradication of residual malignantblasts in patients with leukemia and other types of cancer and inadoptive cellular immunotherapy of cancer and untreatable vital diseasesin allogeneic (MHC-mismatched) patients.

According to this invention, the TALL-104 cells are modified to permitthem to display an increased cytotoxicity against tumor andvirus-infected targets. One modification step includes in vitrotreatment of the TALL-104 cells with a selected cytokine or combinationof cytokines. For example, the two interleukins, rhIL-2 and rhIL-12,when used independently to treat the cell line, induce the cell line'scytotoxic activity. When these cytokines are used together to modify thecell line, the modified cell line displays additive or increasedcytotoxic effects. This results in a significant increase in cytotoxicactivity and recycling capability, ultimately leading to 100%elimination of tumor targets at an E:T ratio<0.1:1 Cesano et al, J.Immunol., 153:2943 (1993)!.

Another modification step of this invention involves the exposure of theTALL-104 cell line to lethal irradiation to confer irreversible loss ofgrowth capability with full retention of cytotoxic activity, both invitro and in vivo. This is achieved by subjecting the cell lines toγ-irradiation just prior to their use. Preferably, the cells areirradiated at 4000 rads using a ¹³⁷ Cs source. The cells may beirradiated for about 30 minutes to achieve this effect.

The modified, lethally irradiated TALL-104 cells are no longerleukemogenic in SCID mice, confirming the gradual loss of viabilityobserved in vitro. The biodistribution and length of survival of theirradiated cells in SCID mouse tissue were investigated by injecting 10⁷TALL-104 cells i.p. into the animals, and by performing PCR analysiswith primers specific to human ALU-DNA sequences on various organs andat different intervals. FIG. 3 compares the kinetics of biodistributionof modified (irradiated) and unmodified (non-irradiated) TALL-104 cellsin the PB, BM, spleen, lung, and liver in mice sacrificed on days 1, 3,and 5 after transfer. On day 1, both modified (irradiated) andunmodified (non-irradiated) TALL-104 cells were present in all of theorgans and tissues examined. On day 3, the irradiated cells were nolonger detectable in the PB and liver; and by day 5, they weredetectable only in the BM. By contrast, as expected from previous work,the non-irradiated cells persisted in every organ (FIG. 3). These datashow the ability of the modified (irradiated) TALL-104 cells tocirculate and persist in SCID mouse tissues, at least for a few daysafter transfer.

Although γ-irradiation is used as an example, it is anticipated thatother methods could be used to stop the growth of the cells, such astreatment with chemical agents that affect DNA synthesis, such asmitomycin C. The in vitro experiments demonstrating the loss ofproliferative activity after irradiation and the persistence ofcytotoxic activity are shown in FIGS. 4A-D and 5A-F. FIG. 4A-D indicatesthat (a) both TALL-104 cells and normal LAK cells lose irreversibly theability to synthesize DNA and RNA within 1 day after γ-irradiation; (b)TALL-104 cells retain their cytotoxic activity after irradiation for atleast 7 days, while normal LAK cells show a steady decline in cytotoxicactivity from day 1 to day 7. FIG. 5A-F shows that the cytotoxicactivity of lethally irradiated TALL-104 cells against three differenttumor targets is similar to that of the non-irradiated counterpartwhereas LAK cell cytotoxicity is highly radiosensitive.

Thus, in contrast to PBL from normal donors and primary leukemic cellsKaufmann, et al, J. Immunol. 139:977-982 (1987)!, both of which do notdisplay cytotoxic activity in response to IL-2 after irradiation, themodified TALL-104 cell line maintains cytotoxic function upon lethalirradiation for at least seven days. Unlike the modified, irradiatedTALL-104 cells, which can still respond very efficiently to activationby IL-2 and/or IL-12, irradiated LAK cells become unresponsive to IL-2and IL-12. The loss of proliferative activity by TALL-104 cells uponirradiation is due to DNA damage and is known to be irreversible.

Therefore, irradiation of TALL-104 cells provides a modified cytotoxiccell line that has lost its proliferative ability and, therefore, thepossibility of growing in an unrestrained fashion in the recipientorganism. In fact, unlike the non-irradiated counterparts, modifiedγ-irradiated TALL-104 cells of this invention transplanted into SCIDmice do not cause leukemia.

On the basis of all the characteristics described above, TALL-104 cellsappropriately modified (i.e., cytokine activated and γ-irradiated) arehighly effective and unable to proliferate and, therefore, suitable foruse in adoptive immunotherapy of cancer and viral diseases inimmunosuppressed HLA-mismatched recipients, as well as for eradicationof residual malignant cells from patient's bone marrow (marrow purging).Unlike the CTL and LAK cells which are being used for adoptive therapyof cancer and viral diseases, the modified TALL-104 cell line is notpatient-specific, but rather may be universally used in treatment of anyHLA mismatched patient.

According to this invention, therefore, modified TALL-104 cells areprepared as follows. TALL-104 cells (ATCC CRL 11386) are exponentiallygrown in tissue culture in the presence of recombinant human (rh) IL-2.Before use, the cells are incubated overnight (i.e., for about 6 hoursor more) in the presence of rh IL-2 (100 U/ml) and/or rh IL-12 (10ng/ml). The cytokine-treated TALL-104 cells are then γ-irradiated usinga Cesium source with gamma rays (at about 4,000 rads). The irradiationmay be continued for a selected time, such as about 30 minutes. Theresulting cell line is referred to as the modified TALL-104 cell line.

The preferred modalities of cancer treatment using the modified TALL-104cells are the following two, as described herein. In one modality, asuitable number of modified TALL-104 cells are administered to arecipient host who receives immunosuppressive treatment with CsAfollowing the criteria used for organ transplantation. Preferably, themode of administration involves injecting the cells i.v. in saline. Itis also anticipated that this treatment may be administered to a patientwho may be already immunosuppressed due to his disease state. Theevaluation of whether the patient's immune status is immunocompetent orseverely compromised may be readily determined by one of skill in theart. Multiple injections of the modified (activated, lethallyirradiated) TALL-104 cells are administered as deemed to be necessary,based on results obtained from preclinical studies with experimentalanimal systems.

The other modality of cancer treatment is marrow purging using themodified (i.e., IL-2/IL-12 activated, γ-irradiated) TALL-104 cells, asdescribed above.

The following experiments demonstrate that the claimed modified cellline can be used effectively and safely as therapeutic agent both inclinical and veterinary practice. These experiments support the utilityof the claimed invention, showing that the modified TALL-104 cell lineis unique, being not patient specific, and endowed with broad anti-tumorand anti-viral cytotoxicity, without being cytotoxic for cells fromnormal tissues. These examples are illustrative only and do not limitthe scope of the present invention.

EXAMPLE 1--TALL-104 CELL LINE

The origin and establishment of the IL-2-dependent TALL-104 cell linefrom the peripheral blood of a T-ALL patient, case CH-23 from theChildren's Hospital of Philadelphia, is described in O'Connor et al,Blood, 77:1534-1545 (1991), incorporated by reference herein. Briefly,mononuclear cells from the leukemic sample were separated by FicollHypaque gradient centrifugation and plated in 24-well Linbro plates FlowLaboratory, McLean, Va.! at a concentration of 1×10⁶ /mL in Iscove'smodified Dulbecco's medium (IMDM) Gibco, Grand Island, N.Y.!supplemented with 10% fetal bovine serum Hycone, Logan, UT! andantibiotics (complete medium). Recombinant human (rh) preparations ofgrowth factors (IL-2, GM-CSF and IL-3) were added at specificconcentrations.

The cells initially proliferated in the presence of IL-2 Amgen!, IL-3and GM-CSF Genetics Institute! and in the absence of added growthfactors. However, after two months in culture, it became apparent thatthe cells in IL-2 proliferated much faster than those in the otherconditions. These cells became established as an IL-2-dependent cellline, designated TALL-104, whereas those maintained in IL-3, GM-CSF, orno factor had a finite life span of about 6 months.

This TALL-104 cell line has a T cell phenotype (CD2, CD3, CD7, CD8,CD56), and has been in continuous culture since 1990 in the laboratoryof the inventor, Dr. Santoli. The TALL-104 cells are maintained at 37°C. in 8%-10% CO₂ in IMDM (Gibco) supplemented with 10% fetal bovineserum. Biweekly addition of fresh medium containing rhIL-2 is requiredfor optimal viability and continuous growth of TALL-104 cells; whenthese cells are propagated in the absence of this factor, they remainviable for a few weeks but lose cytotoxic activity gradually within amonth Cesano and Santoli, In Vitro Cell. Dev. Biol., 28A:648-656(1992)!.

EXAMPLE 2--CYTOTOXICITY OF TALL-104 Cells

The MHC non-restricted tumoricidal activity of the IL-2 dependentTALL-104 cell line has been studied in detail, using standard ⁵¹Cr-release assays, as described Cesano et al, Blood, 77:2463-2474(1991); O'Connor et al, Blood, 77:1534-1545 (1991) and Cesano andSantoli, In Vitro Cell, Dev. Biol., 28A:648-656 (1992)!, incorporated byreference herein.

Prior to the assay, the effector cells are incubated in rhIL-2.IL-2-stimulated cells kill very efficiently a large variety of tumorcell lines and freshly separated leukemic samples but not cells fromnormal tissues (see Tables II and III). The TALL-104 cells can also betriggered by CD2- and CD3-specific monoclonal antibodies (mAb) tomediate cytotoxic activity against FcR⁺ tumor cell lines Cesano andSantoli, In Vitro Cell. Dev. Biol., 28A:648-656 (1992)!. In addition,this cell line produces high levels of IFN-γ, TNF-α, TGF-β1 and GM-CSFon stimulation with antibodies recognizing CD2, CD3 and other surfacemolecules, tumor cells, IL-2, and IL-12 Cesano and Santoli, In VitroCell. Dev. Biol., 28A:657-662 (1992); Cesano et al, J. Immunol.,151:2943 (1993)!, incorporated by reference herein.

EXAMPLE 3--LACK OF CYTOTOXICITY OF TALL-104 CELL LINE AGAINST CELLS FROMNORMAL TISSUES

The TALL-104 cell line, obtained as described herein, is not cytotoxicfor cells from normal tissues and, therefore, can be used safely both exvivo (marrow purging) and in vivo (adoptive transfer) because normalcells would not be killed. Failure of TALL-104 cells to kill cells fromnormal tissues is due to their inability to recognize and formconjugates with such targets.

Because both unirradiated and modified (irradiated) TALL-104 cellsproduce high levels of TNF-α and IFN-γ upon stimulation with IL-2 andIL-12 and/or exposure to tumor targets Cesano and Santoli, In Vitro CellDev. Biol., 28A:657-662 (1992b)!, 2-week clonogenic assays wereperformed to exclude the possibility that such toxic lymphokinesreleased by TALL-104 cells would impair the growth of hematopoieticprecursors in bone marrow samples. FIG. 7A-D shows that in bone marrowsadmixed with TALL-104 cells at 0.1 and 1:1 ratios, the clonogenic growthof committed progenitors (CFU, BFU) was very similar to that of marrowsincubated without TALL-104 cells.

Because the pluripotent CD34⁺ stem cells are indispensable for asuccessful hematopoietic reconstitution, the number of CD34⁺ cells wasevaluated in T-cell depleted bone marrow samples before and after an 18hour incubation with modified (γ-irradiated) TALL-104 cells. Asdetermined by immunofluorescence analysis, the percentage of CD34⁺ cellsremained the same in the bone marrow samples incubated with TALL-104cells (6-8% and 5.5-7.5% CD34⁺ cells were found in untreated andTALL-104 treated bone marrow, respectively). Moreover, thecolony-forming activity of CD34 cell-enriched cord blood samples wasfound to be totally unaffected upon an 18-hour incubation with lethallyirradiated TALL-104 cells. All together, these results indicate thatmodified TALL-104 cells do not display any toxicity on normal stem cellsnor on lineage-committed precursors and could, therefore, be used safelyfor bone marrow purging against leukemia.

EXAMPLE 4--EFFICACY OF MODIFIED (LETHALLY IRRADIATED) TALL-104 CELLS INMARROW PURGING

To test the efficacy of modified, i.e., lethally irradiated, TALL-104cells in marrow purging, the leukemia cell line U937 tagged with theneomycin-resistance gene was admixed at various ratios (0-50%) withcells from normal bone marrow, and modified, i.e., IL-2/IL-12 activated,γ-irradiated, TALL-104 cells were added at the ratio of 1:1 relative tobone marrow cells. The cell mixtures were then incubated for 18 hoursand subjected to colony assays. No U937 cells could be detectedmicroscopically after a 2-week culture in methylcellulose (Table V). PCRamplification of the Neomycin resistance gene 18 hours after theaddition of TALL-104 cells to bone marrow/U937 samples confirmed thetotal absence of U937 cells from such samples at all E/T ratios used(FIG. 8).

                  TABLE V                                                         ______________________________________                                                      % U937 Cells in the                                                                        Number of U937                                     TALL-104/BM Ratio                                                                           Bone Marrow  Colonies                                           ______________________________________                                        0/1            0           0                                                  0/1            5           19                                                 0/1           10           40                                                 0/1           25           112                                                0/1           50           235                                                1/1            0           0                                                  1/1            5           0                                                  1/1           10           0                                                  1/1           25           0                                                  1/1           50           0                                                  ______________________________________                                    

In conclusion, these studies (Examples 3 and 4) indicate that modified,i.e., lethally irradiated, TALL-104 cells represent a unique cell systemto design clinically safe and highly effective marrow purging strategiesfor future ABMT programs. Moreover, this purging strategy for theelimination of MRD in leukemia is applicable to any form of cancerdisplaying bone marrow involvement, including neuroblastoma in childrenand breast carcinoma in adult patients.

EXAMPLE 5--ANTITUMOR EFFICACY OF MODIFIED TALL-104 CELLS IN ADOPTIVEIMMUNOTHERAPY

The following experiments carried out in experimental animals show thatthe modified TALL-104 cells of this invention can be used effectively inadoptive immunotherapy treatment of cancer. These treatments aredemonstrated both in severe combined immunodeficient (SCID) mice, whichis an animal model for cancer treatment accepted by one of skill in theart, and in immunocompetent mice treated with CsA to prevent allograftrejection.

For use in the following experiments, the TALL cells of the inventionwere modified as follows. The cells were exponentially grown in tissueculture in the presence of IL-2. Before use, the cells were washed,incubated overnight in the presence of IL-2 (100 U/ml) and/or IL-12 andγ-irradiated using a Cesium source with gamma rays (4000 rads).

A. Prevention and regression of acute myelogenous leukemia (AML) in SCIDmice

SCID mice were implanted i.p. with the human myeloid leukemia cell lineU937 ATCC CRL 1593!. These cells are very aggressive and kill mice in 21days if injected i.v. Cesano et al, Oncogene, 7:827 (1992)! or only 8-10days if injected i.p. Treatment with modified TALL-104 cells was done atdifferent times after injection of the AML target, and the survival ofthe mice was determined for a span of several months, as indicated inFIG. 9. Survivors, showing no symptoms after a 6-month observationperiod, were sacrificed and the presence of human cells (both effectorsand targets) in the BM was evaluated by PCR analysis using primersspecific for human ALU-DNA sequences. The most remarkable finding in theexperiments presented in FIG. 9 is that multiple transfers, at shortintervals, of modified, IL-2-activated TALL-104 cells cured 50% of themice from an advanced stage of leukemia. In fact, of the mice injectedwith TALL-104 cells three times starting on day 3, when U937 cells hadalready infiltrated the PB, two developed AML and died 3 months later,and two remained clinically and morphologically free of disease for atleast 6 months (FIG. 9). PCR amplification of human ALU-DNA sequencesprovided molecular evidence for the persistence of residual cells in theBM of the survivors. Whether these cells represented TALL-104 effectorsor U937 targets could not be determined in this set of experiments.Importantly, complete abrogation of AML, as monitored both clinicallyand by PCR analysis, was achieved by repeated injection of the effectorcells on days 0, 3, and 6 (FIG. 9).

When the modified TALL-104 cells were injected at the same time as U937cells on day 0 (i.e., without any delay) but on the opposite flank, PCRamplification of human ALU-DNA sequences in the BM of these mice,electively sacrificed 6 months from treatment, demonstrated that bothtypes of leukemic populations, the myeloid target and the lymphoideffector, were eliminated from the mouse tissues. Another interestingobservation from the experiments shown in FIG. 9 is that administrationof low doses of rh IL-12 (1 μg/d for 7 d) in conjunction with TALL-104cells at the time of U937 cell challenge induced a significantly longersurvival in the animals, as compared to mice receiving U937 and modified(IL-2 activated) TALL-104 cells alone, without rh IL-12. Overall, thefindings in FIG. 9 indicate that, depending on the tumor load at thetime of treatment, adoptive transfer of modified TALL-104 cells eitherinduced complete abrogation of AML or inhibited significantly tumorgrowth even during advanced stages of the disease. Thus, the extent oftumor reduction appears to reflect the E/T ratio at the time oftreatment.

Based on the observations that lethally irradiated TALL-104 cellsexpress tumoricidal activity in vitro (FIGS. 4A-D and 5A-F) and are ableto circulate in SCID mouse tissues (FIG. 3), experiments were performedto determine the extent to which TALL-104 cells modified byγ-irradiation display anti-tumor effects in the SCID/U937 model. To havean objective way for detection of MRD in the treated mice, the U937clone tagged with the neomycin-resistance gene was used in theseexperiments. FIGS. 10A and 10B show the survival curve and PCR data,respectively, from an experiment in which SCID mice were challenged withNeo⁺ U937 cells and treated, or not, with the modified irradiatedTALL-104 cells. The effectors were transferred on alternate days (2×10⁷,i.p.), starting from the day of U937 cell challenge: as also indicatedin the experiment shown in FIG. 9, TALL-104 cells were co-injected withU937 cells on day 0 in the opposite flank. Modified TALL-104 cells weretransferred either alone or in conjunction with rh IL-2 (100 &/d) or rhIL-12 (1 μg/d), daily for 7 days. All mice receiving only U937 cells(107 i.p.), either alone or in combination with daily administration ofIL-2 and IL-12 for 1 week, died between days 11 and 16 (FIG. 10A). Incontrast, mice treated with modified irradiated TALL-104 cells, alone orin conjunction with the cytokines, remained disease-free (lookedclinically well and showed no signs of disease) for at least 2 months(FIG. 10A). At this time, PCR amplification of human ALU-DNA sequencesdemonstrated the presence of human cells in the BM of mice treated withmodified TALL-104 cells alone or treated with modified TALL-104 cellsand rh IL-12 (FIG. 10B). PCR analysis using neomycin-specific primersconfirmed the presence of residual U937 cells in the same samples (FIG.10B). By contrast, administration of rh IL-2 to modified TALL-104cell-treated mice resulted in the total disappearance of molecularlydetectable leukemic cells, using either type of primers (FIG. 10B).These data indicate that, in vivo, the modified γ-irradiated TALL-104cells are still responsive to low doses of rh IL-2, whereas higher dosesof rh IL-12 may be desirable.

B. Inhibition of human glioblastoma growth in SCID mice

SCID mice were engrafted subcutaneously with the human U-87 MGglioblastoma cell line ATCC HTB 14! using conventional techniques. Thiscell line induces a local tumor, ultimately resulting in skin ulcerationwithin 4 weeks.

The modified (IL-2 or IL-12 activated, γ-irradiated) TALL-104 cells wereadoptively transferred to the SCID mice by injection at the tumor site.Control animals received only PBS. For the smaller tumor (2 mm), therapywas applied by local injection 3×, every 10 days. For the larger tumor(5 mm), therapy was applied by local injection 5×, on alternate days. Inanother set of experiments, the irradiated killers were transferredthree times on alternate days starting from the day of U87 MG cellchallenge.

The results are provided in Table VI. Column 1 provides the tumor sizeat the time of modified TALL-104 cell transfer. Column 2 indicateswhether modified TALL-104 cells or just PBS were administered. Column 3provides the tumor load at the time the mice were sacrificed. Column 4provides the percentage of tumor reduction in modified TALL-104-injectedanimals as compared to control mice receiving only PBS.

                  TABLE VI                                                        ______________________________________                                        Reduction of subcutaneous growth of a human glioblastoma                      in SCID mice upon adoptive transfer of modified (γ-                     irradiated and IL-2 or IL-12 treated) TALL-104 cells.                                                           % Tumor                                     Tumor size (mm)                                                                           Therapy   Tumor load (mg)                                                                           Reduction                                   ______________________________________                                        2           PBS       .sup. 662 ± 40.sup.a                                                                   ˜75%                                              TALL-104  225 ± 40                                             5           PBS       1062 ± 103                                                                             ˜50%                                              TALL-104  535 ± 110                                            0           PBS       784 ± 228                                                                               100%                                                   TALL-104  0                                                       ______________________________________                                         .sup.a Mean ± standard deviation from three mice.                     

As shown in the table above, mice injected at the tumor site withmodified (cytokine treated and γ-irradiated) TALL-104 cells displayed a50-100% reduction of tumor load (as compared to control mice receivingonly PBS), depending on the size of the original tumor when therapy wasstarted.

C. Treatment of cyclosporin A-treated immunocompetent mice bearingsyngeneic leukemia

To investigate the feasibility of using modified (lethally irradiated)TALL-104 cells of the invention in MHC incompatible, immunocompetentrecipients, the following experiment was conducted.

TALL-104 cells were obtained as described in Example 1 grown in tissueculture in the presence of IL-2 and then modified by γ-irradiation asfollows. The cells were washed in 50 ml of IMDM medium, incubated at 37°C. overnight in the presence of 100 U/ml IL-2 and 10 ng/ml IL-12, andirradiated using a Cesium source with gamma rays (4000 rads).

10⁷ cells of a murine B lymphoid leukemia cell line (70z) were engraftedintraperitoneally into syngeneic immunocompetent three week old malemice (DBA/2) using conventional techniques. The mice were randomlyassigned to three groups, five per group.

In a first group, the mice were treated from the day of implant of 70zcells (day 0) with multiple intraperitoneal (i.p.) transfers of 2×10⁷ ofthe modified irradiated TALL-104 cells on alternate days for 10 days inconjunction with daily i.p. administration of 10 mg/kg cyclosporin A(CsA) once a day. Neither IL-12 nor IL-2 were administered to the mice.

In a second group (control), the leukemia-bearing mice were treated onlywith CsA for 10 days. No TALL-104 cells were introduced into these mice.

In a third group (control), the leukemia-bearing mice were treated withmultiple transfers of modified irradiated TALL-104 cells on alternatedays for 10 days, as described above, with the exception that no CsA wasadministered.

All five mice treated with multiple transfers of modified irradiatedTALL-104 cells on alternate days for 10 days in conjunction with dailyadministration of CsA were clinically cured (group 1). PCR analysisperformed 3 months later on all five asymptomatic mice providedmolecular evidence for the absence of 70z leukemia in their tissues. Incontrast, both control groups of leukemia-bearing mice treated only withCsA (without modified TALL-104 cells) or only with modified TALL-104cells (without CsA), died of leukemia within 12 days (groups 2 and 3).

This data indicates that modified (lethally irradiated) TALL-104 cellsare not rejected and function effectively in immunocompetent recipientsreceiving immunosupressive treatment (CsA). The fact that the TALL-104treated mice remained healthy for at least 3 months after treatmentindicates that the TALL-104 cells do not proliferate in the mice and donot cause toxic effects.

EXAMPLE 6--ABILITY OF MODIFIED (LETHALLY IRRADIATED) TALL-104 CELLS TOKILL DOG LYMPHOMAS AND CAT LEUKEMIAS

Four cell lines established from either dog lymphomas (Pilgrim andTertiat's) or cat leukemias (CAT 3281 and CAT 3201B) were analyzed forsusceptibility to TALL-104 cell killing in a ⁵¹ Cr-release assay. FIG.11A and B shows that all four cell lines are highly susceptible toTALL-104 lysis within 4 hours from the interaction with the effectorcells (top panel) and are killed 80-100% within 18 hours (bottom panel)even at low effector/target ratios. FIG. 12A and B shows that modified,lethally irradiated TALL-104 cells kill these targets with an efficiencysimilar to the unirradiated cells as measured both in a 4-hour (top) and18-hour (bottom) ⁵¹ Cr-release assay.

Because all four of the above cell lines can be engrafted in SCID mousetissues, studies are being performed to analyze the ability of modifiedlethally irradiated TALL-104 cells to control the growth of catleukemias and dog lymphomas in these chimeric SCID models. Resultsindicated that, indeed, modified TALL-104 cells can, in appropriateexperimental conditions, cure SCID mice bearing such tumors. It isanticipated that studies in dogs and cats will reveal the safety of theuse of modified irradiated TALL-104 cells in veterinary practice.

The cell line of this invention has been deposited in the culturecollection of the American Type Culture Collection, in Rockville, Md. onJun. 15, 1993, and identified there as "TALL-104". The cell line wastested and determined to be viable on Jun. 18, 1993. This culture wasassigned ATCC Accession No. CRL 11386. The ATCC has agreed to maintainthe culture for a period of 30 years from deposit date, or until atleast five years after the most recent request for a sample, whicheveris longer.

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. Such modifications and alterationsto the methods of the present invention are believed to be encompassedin the scope of the claims appended hereto.

What is claimed is:
 1. Modified TALL-104 cells ATCC No. CRL 11386characterized by cytotoxic activity in vitro and in vivo, irreversiblyarrested cell proliferation, and the capacity to migrate in vivo todifferent tissue, wherein the TALL-104 cells are stimulated in vitro bytreatment with one or a combination of the cytokines selected from thegroup consisting of IL-2 and IL-12 to enhance the cytotoxic activity ofsaid TALL-104 cells against malignant cells, and γ-irradiating the cellsat a dose suitable to irreversibly arrest cell proliferation but notinterfere with the cytotoxic activity in vitro and in vivo of thestimulated TALL-104 cells.
 2. The γ-irradiated modified TALL-104 cellsaccording to claim 1, wherein said suitable dose is about 4000 rads. 3.A therapeutic reagent comprising the modified TALL-104 cells accordingto claim 1 in a suitable pharmaceutical carrier.
 4. A method forpreparing a therapeutic reagent comprising stimulating TALL-104 cellsATCC No. CRL 11386 in vitro by treating said cells with at least one ora combination of two cytokines selected from the group consisting ofIL-2 and IL-12 to enhance the cytotoxic activity of said cells againsttumor and virus-infected cells, and γ-irradiating said TALL-104 cells ata dose suitable to irreversibly arrest cell proliferation, wherein saidγ-irradiated TALL-104 cells retain their cytotoxic activity in vitro andin vivo.
 5. The method according to claim 4 wherein said suitable doseis about 4000 rads.
 6. Modified TALL-104 cells prepared by the method ofclaim
 4. 7. A therapeutic reagent comprising modified TALL-104 cellsprepared by the method of claim 4 in a suitable pharmaceutical carrier.8. A veterinary composition comprising modified TALL-104 cells preparedaccording to the method of claim 4 in a suitable veterinary carrier. 9.A preparation of proliferation-arrested TALL-104 cell, wherein TALL-104cells ATCC No. CRL 11386 are γ-irradiated at a dose suitable toirreversible arrest cell proliferation but not interfere with cytotoxicactivity of the cells against tumor and virus infected cells, whereinsaid TALL 104 cells have been previously stimulated by treatment withone or a combination of the cytokines selected from the group consistingof IL-2 and IL-12.
 10. The preparation according to claim 9, whereinsaid cells exhibit cytotoxicity to tumor cells.